Abstract
The Ppz enzymes are Ser/Thr protein phosphatases present only in fungi that are characterized by a highly conserved C-terminal catalytic region, related to PP1c phosphatases, and a more divergent N-terminal extension. In Saccharomyces cerevisiae, Ppz phosphatases are encoded by two paralog genes, PPZ1 and PPZ2. Ppz1 is the most toxic protein when overexpressed in budding yeast, halting cell proliferation, and this effect requires its phosphatase activity. We show here that, in spite of their conserved catalytic domain, Ppz2 was not toxic when tested under the same conditions as Ppz1, albeit Ppz2 levels were somewhat lower. Remarkably, a hybrid protein composed of the N-terminal extension of Ppz1 and the catalytic domain of Ppz2 was as toxic as Ppz1, even if its expression level was comparable to that of Ppz2. Similar amounts of yeast PP1c (Glc7) produced an intermediate effect on growth. Mutation of the Ppz1 myristoylable Gly2 to Ala avoided the localization of the phosphatase at the cell periphery but only slightly attenuated its toxicity. Therefore, the N-terminal extension of Ppz1 plays a key role in defining Ppz1 toxicity. This region is predicted to be intrinsically disordered and contains several putative folding-upon-binding regions which are absent in Ppz2 and might be relevant for toxicity.
Highlights
The Saccharomyces cerevisiae Ppz1 protein phosphatase is a 692-residue protein made of a C-terminal catalytic domain, about 60% identical to Glc7, the catalytic subunit of genuine yeast PP1 (PP1c), and a long N-terminal segment (~350 residues) unrelated to other proteins [1]
In S. cerevisiae, this is done by downregulating the influx of potassium through the high-affinity Trk transporters and by repressing the expression of the Na+/K+-ATPase encoded by the ENA1 gene [11,12,13,14]
The overexpression of a chimeric protein bearing the catalytic domain of Ppz2 and the N-terminal extension of Ppz1 was as toxic as that of Ppz1, even if its expression level was similar to that of Ppz2. These results indicate that the N-terminal extension of Ppz1 might have a role in the toxicity of the phosphatase that is not shared by the equivalent N-terminal half of Ppz2
Summary
The Saccharomyces cerevisiae Ppz protein phosphatase is a 692-residue protein made of a C-terminal catalytic domain, about 60% identical to Glc, the catalytic subunit of genuine yeast PP1 (PP1c), and a long N-terminal segment (~350 residues) unrelated to other proteins [1]. Cells lacking Ppz are hypertolerant to Na+ and Li+ cations and sensitive to conditions or drugs affecting cell wall integrity, such as high temperature or treatment with calcofluor white or caffeine [2,3,12,15,16] The latter effect has been explained by the increased turgor pressure caused by augmented K+ influx in ppz cells [16]. The overexpression of a chimeric protein bearing the catalytic domain of Ppz and the N-terminal extension of Ppz was as toxic as that of Ppz, even if its expression level was similar to that of Ppz2
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